Winkler Barry S, Pourcho Roberta G, Starnes Catherine, Slocum Jessica, Slocum Nicklaus
Eye Research Institute, Oakland University, 406 Dodge Hall, Rochester, MI 48309, USA.
Exp Eye Res. 2003 Sep;77(3):327-37. doi: 10.1016/s0014-4835(03)00147-7.
It has long been known that mammalian retinas metabolize glucose aerobically to lactic acid and carbon dioxide. The classical view holds that glucose is the primary substrate for energy metabolism in all retinal cells, and that photoreceptor cells have the highest rates of glycolysis and respiration. A different and more recent view is that the Müller cells are the principal, if not sole aerobic producers of lactate, which then serves as the primary fuel for the mitochondria in photoreceptor cells and other retinal neurons. In this paper, we have examined these two competing hypotheses in rat and guinea pig retinas by identifying the cellular sites of glucose uptake and phosphorylation via hexokinase by means of autoradiographic studies with 3H-2-deoxyglucose (3H-2DG). The rat retina serves as a vascular model and the guinea pig retina serves as an avascular model. Rat and guinea pig eyecups were incubated in oxygenated, bicarbonate-buffered media containing glucose in the presence of labeled and unlabeled 2DG. Biochemical measurements of lactate production and ATP content were made on rat retinas incubated with different concentrations of glucose and 2DG in order to establish the optimal condition for conducting the autoradiographic studies with 3H-2DG. The optimal substrate concentrations were 1mM glucose and 0.25 mM 2DG. Results showed that following incubation of dark-adapted rat eyecups for 1 hr in media containing 1mM glucose/0.25 mM 2DG and supplemented with 3H-2DG, the label was distributed throughout all the layers of the retina, from the ganglion cell layer to the retinal pigment epithelium, with denser label associated with the outer retina (photoreceptors) relative to the density of label in the inner retina, as evaluated by counts of silver grains in individual retinal layers. Exposure of rat eyecups to light did not alter the relative distribution of label, but did increase total grain counts by 70%. However, uptake of labeled 2DG, as measured by scintillation counting of radioactivity in trichloroacetic acid extracts, was not significantly different between light- and dark-adapted rat retinas. In guinea pig eyecups, labeled 2DG was distributed throughout all the retinal layers. Addition of 10mM lactate or pyruvate to the glucose/2DG media produced no measurable change in the density or distribution of label in the eyecups. Measurements of the activity of hexokinase in rat retinas revealed that this enzyme was present in both the mitochondrial and cytosolic fractions. The present results suggest that as long as the availability of ambient glucose is adequate, retinal neurons use glucose, rather than glial-derived lactate, as the major substrate for the production of high energy phosphates.
长期以来，人们一直知道哺乳动物的视网膜会将葡萄糖有氧代谢为乳酸和二氧化碳。传统观点认为，葡萄糖是所有视网膜细胞能量代谢的主要底物，且光感受器细胞的糖酵解和呼吸速率最高。一种不同的、更新的观点是，米勒细胞是乳酸的主要（即便不是唯一的）有氧产生者，而乳酸随后作为光感受器细胞和其他视网膜神经元中线粒体的主要燃料。在本文中，我们通过使用³H-2-脱氧葡萄糖（³H-2DG）进行放射自显影研究，确定葡萄糖摄取和通过己糖激酶磷酸化的细胞位点，从而在大鼠和豚鼠视网膜中检验了这两种相互竞争的假说。大鼠视网膜作为血管模型，豚鼠视网膜作为无血管模型。将大鼠和豚鼠的眼球杯在含有葡萄糖的充氧、碳酸氢盐缓冲培养基中孵育，培养基中同时存在标记和未标记的2DG。对用不同浓度的葡萄糖和2DG孵育的大鼠视网膜进行乳酸产生和ATP含量的生化测量，以便确定进行³H-2DG放射自显影研究的最佳条件。最佳底物浓度为1mM葡萄糖和0.25mM 2DG。结果显示，在含有1mM葡萄糖/0.25mM 2DG并补充³H-2DG的培养基中，将暗适应的大鼠眼球杯孵育1小时后，标记物分布在视网膜的所有层，从神经节细胞层到视网膜色素上皮，相对于内视网膜中标记物的密度，外视网膜（光感受器）的标记物更密集，这通过对各个视网膜层中银颗粒的计数来评估。将大鼠眼球杯暴露于光下并没有改变标记物的相对分布，但确实使总颗粒计数增加了70%。然而，通过三氯乙酸提取物中放射性的闪烁计数测量，标记的2DG摄取在光适应和暗适应的大鼠视网膜之间没有显著差异。在豚鼠眼球杯中，标记的2DG分布在所有视网膜层。向葡萄糖/2DG培养基中添加10mM乳酸或丙酮酸，眼球杯中标记物的密度或分布没有产生可测量的变化。对大鼠视网膜中己糖激酶活性的测量表明，这种酶存在于线粒体和胞质部分。目前的结果表明，只要环境葡萄糖的可用性充足，视网膜神经元就使用葡萄糖而非神经胶质来源的乳酸作为产生高能磷酸盐的主要底物。
